Affinity chromatography allows for the purification of a protein of interest from a mixture of molecules, such as a cellular harvest, based on the preferential binding of the protein of interest to a target in solid phase, such as a gel matrix. This solid phase component typically is formed into a column through which the mixture containing the protein of interest is applied. In this initial step, called the capture step, the protein of interest specifically binds to the target in solid phase whereas other components in the mixture flow through the column. However, certain components within the mixture, including high molecular weight species (HMWs), low molecular weight species (LMWs) and host cell proteins (HCPs), may remain within the column as impurities along with the protein of interest. Thus, typically one or more wash steps are carried out in which one or more wash solutions are applied to the column to remove these impurities while maintaining the binding of the protein of interest to the solid phase. Finally, after removal of impurities by the washing step(s), the protein of interest is recovered from the column by an elution step, in which an elution solution that disrupts the binding of the protein of interest to the solid phase target, is applied to the column and the protein of interest is recovered in the eluate. Accordingly, the effectiveness of affinity chromatography in purifying a protein of interest depends in large part on identifying wash conditions that allow for efficient removal of impurities (e.g., HMWs, LMWs, HCPs) while not disrupting the binding of the protein of interest to the solid phase target or otherwise having undesired effects.
A particularly useful type of affinity chromatography is Protein A chromatography for the purification of proteins that contain an immunoglobulin Fc region, such as antibodies and Fc fusion proteins. Various wash solutions have been described for removal of impurities from Protein A columns, including wash solutions containing one of the following: hydrophobic electrolytes (e.g., tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride or tetrabutylammonium chloride at pH1-5.0-7.0), solvents (e.g., 5-20% isopropanol or polypropylene/hexylene glycol), urea (e.g., at a concentration of 1-4 M), detergents (e.g., 0.1-1% Tween 20 or Tween 80), polymers (e.g., 5-15% polyethylene glycol such as PEG400 or PEG8000) or highly concentrated buffer solutions such as Tris, HCl, acetate, sulfate, phosphate or citrate buffers at a concentration of 0.8-2.0 M at a pH between 5.0 and 7.0 (see e.g., Shukla, A. A. and Hinckley, P. (2005) Biotechnol. Prog. 24:1115-1121; U.S. Pat. Nos. 6,127,526 and 6,333,398 by Blank; and U.S. Pat. No. 6,870,034 by Breece et al.). Many of these chemicals, however, have one or more disadvantages, including but not limited to toxicity, corrosiveness, flammability, instability, costly disposal as hazardous waste and/or inefficient removal of contaminants during the washing step.
Protein A chromatography wash buffers containing salt (such as sodium chloride), alone or in combination with either a detergent (e.g. Tween 20), a solvent (e.g., hexylene glycol) or a polymer (e.g., polyethylene glycol), have also been described (U.S. Pat. No. 6,870,034 by Breece et al.).
Barron et al. describe an intermediate wash solution for Protein A chromatography containing 0.5 to 2.0 M arginine in a phosphate/acetate buffer at pH 5.0-7.5 (optimally 1M arginine, 0.1M phosphate/acetate buffer at pH 5.0). This arginine wash step is reported to remove HCP contaminants. The authors also tested an intermediate wash solution that contained sodium chloride at 0.5-2.0 M at pH 5.0-7.5 but reported that the NaCl wash showed no significant decrease in HCP (Barron et al., “Improving Purity on Protein A Affinity Media Through Use of an Arginine Intermediate Wash Step”, http://www.priorartdatabase.com/IPCOM/000127319).
Sun et al. also describe washing of affinity chromatography columns, such as a Protein A column, with a wash buffer that contains arginine, or an arginine derivative, at a concentration of 0.1-2.0 M and at a pH of 4.5-8.0 (U.S. Patent Publication Nos. 20080064860 and 20080064861; PCT Publication No. WO 2008/031020).
Arginine has also been used to elute proteins from affinity chromatography columns and other types of purification columns. For example, Arakawa et al. describe methods of eluting antibodies from a Protein A column using an elution buffer containing 0.5-20 M arginine at pH 4.1-5.0 (Arakawa et al. (2004) Protein Expression and Purification 36:244-248; Tsumoto, K, et al. (2004) Biotechnol. Prog. 20:1301-1308; U.S. Patent Publication No. 20050176109). Additionally. U.S. Pat. No. 7,501,495 by Ejima et al. describes methods of eluting proteins from a gel filtration column by using a developing solution containing arginine hydrochloride. Ghose et al. describe methods of eluting proteins of interest from underivatized silica using an arginine gradient as the eluant (Ghose, S. et al. (2004) Biotech. Bioeng. 87:413-423). U.S. Patent Publication No. 20030050450 by Coffman et al. describes methods of dissociating Fc containing molecules from complexes of the Fe containing molecule and Protein A, wherein the Fc/Protein A complexes are applied to a hydrophobic interaction column (HIC) and the column is washed with a buffer containing arginine.